Co-purification of Tat Complex Components

For co-purification of TatC_HIS, TatB and SufI_FLAG, cultures of DADE-P carrying either pFAT75ΔA-sufI_FLAG (control plasmid) or pBC_HIS-sufI_FLAG derivatives were grown as for membrane fraction preparation, but in the presence of 1 mM IPTG. After sonication, clarified cell lysates were solubilized overnight with 1.5 % digitonin in Buffer 1 (20 mM Tris-HCl, pH7.5, 200 mM NaCl, 50 mM imidazole, 10 % glycerol) and the soluble fraction separated by ultracentrifugation. TatC_HIS complexes with TatB and SufI_FLAG were captured with Ni-NTA magnetic beads (ThermoFisher), which were washed three times with wash buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 25 mM imidazole, 0.5 % digitonin) before elution with 1× SDS-PAGE loading buffer (Brand) by mild thermal treatment (53 °C for 10 min). Samples were then run on SDS-PAGE gradient gels (BioRad) and analysed by Western blotting with anti-TatB [15 (link)], anti-His (Invitrogen) or anti-FLAG (Sigma) antibodies. Blots were developed with ECL (BioRad).